Molecular Phylogenetics and Evolution 100 (2016) 51–56
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Short Communication
Back to solitude: Solving the phylogenetic position of the Diazonidae
using molecular and developmental characters
Noa Shenkar a,b,⇑, Gil Koplovitz a,b,c,d,1, Liran Dray a,1, Carmela Gissi e, Dorothée Huchon a,b
a
Department of Zoology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 6997801, Israel
Steinhardt Museum of Natural History and Israel National Center for Biodiversity Studies, Tel-Aviv University, Tel-Aviv 6997801, Israel
c
Eilat Campus, Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel
d
The Inter-University Institute for Marine Sciences of Eilat, Eilat, Israel
e
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari ‘‘Aldo Moro”, Bari, Italy
b
a r t i c l e
i n f o
Article history:
Received 29 September 2015
Revised 27 March 2016
Accepted 1 April 2016
Available online 2 April 2016
Keywords:
Aplousobranchia
Rhopalaea
Mitochondrial genome
Mitogenomics
Phylogeny
Thorax-regeneration
a b s t r a c t
The order Aplousobranchia (Chordata, Ascidiacea) contains approximately 1500 species distributed
worldwide. Their phylogeny, however, remains unclear, with unresolved family relationships. While
most Aplousobranchia are colonial, debates exist concerning the phylogenetic position of families such
as the Diazonidae and Cionidae, which exhibit a solitary lifestyle and share morphological characteristics
with both Aplousobranchia and Phlebobranchia orders. To clarify the phylogenetic position of the
Diazonidae and Cionidae, we determined the complete mitochondrial sequence of the solitary diazonid
Rhopalaea idoneta. The phylogenetic reconstruction based on the 13 mitochondrial protein coding genes
strongly supports a positioning of Diazonidae well-nested within the Aplousobranchia rather than a
positioning as a sister clade of the Aplousobranchia. In addition, we examined the regenerative ability
of R. idoneta. Similar to colonial Aplousobranchia, R. idoneta was found to be able to completely regenerate its thorax. Ciona, also known to possess high regenerative abilities, is the Aplousobranchia sister clade
rather than a member of the Phlebobranchia. Our results thus indicate that the colonial lifestyle was
acquired in the Aplousobranchia, starting from a Ciona-like solitary ancestor and secondarily lost in
Diazonidae representatives such as Rhopalaea. The solitary lifestyle of Rhopalaea is thus a derived
characteristic rather than an ancestral trait.
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
The class Ascidiacea (Phylum: Chordata) is the largest and most
diverse class of the sub-phylum Tunicata (also known as Urochordata). With nearly 3000 valid species, which include important
model organisms such as Ciona intestinalis and Botryllus schlosseri
(Dehal et al., 2002; Voskoboynik et al., 2013), this unique group
of invertebrates provides developmental and evolutionary biologists with important insights into evolutionary events at the origin
of the vertebrate subphylum. However, phylogenetic relationships
between ascidian orders and families, in particular within the
Aplousobranchia, as well as the validity of certain lineages, remain
to be ascertained.
Nowadays, the majority of phylogenetic studies follow Lahille’s
(1887) classification of the class Ascidiacea into three orders based
⇑ Corresponding author at: Department of Zoology, George S. Wise Faculty of Life
Sciences, Tel-Aviv University, Tel-Aviv 6997801, Israel.
E-mail address: [email protected] (N. Shenkar).
1
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ympev.2016.04.001
1055-7903/Ó 2016 Elsevier Inc. All rights reserved.
on the structure of the adult branchial sac: the order Aplousobranchia with the simplest branchial sac (Aplouso = simple in
Greek); the order Phlebobranchia (Phlebo = vessel in Greek), which
possesses distinct longitudinal blood vessels in its branchial sac;
and the order Stolidobranchia, which is distinguished by the
folding of the branchial wall (Stolido = folded, pleated in Greek)
(Monniot et al., 1991). Both colonial and solitary lifestyles are
reported among all three orders, depending on the particular taxonomic approach considered as valid (Moreno and Rocha, 2008).
For example, the phylogenetic position of the solitary Ciona intestinalis has been a matter of controversy, as its large branchial sac and
its internal longitudinal branchial vessels are typical of solitary
phlebobranchs (Monniot, 1991); while its vanadium oxidation
state (Hawkins et al., 1983) and the regenerative role of its
epicardial tissue imply a closer relationship with aplousobranchs
(Kott, 1990).
The family Diazonidae comprises both solitary (i.e., Rhopalaea)
and colonial (i.e., Diazona) species, and shares some primitive characters with the family Cionidae and other Phlebobranchia, such as
numerous inner longitudinal branchial vessels, a relatively large
52
N. Shenkar et al. / Molecular Phylogenetics and Evolution 100 (2016) 51–56
branchial sac, and a gelatinous translucent test (Kott, 1990). However, the Diazonidae also have Aplousobranchia characteristics:
while in solitary phlebobranch species the gut loop is found behind
the pharynx with no constriction of the body wall separating the
abdomen from the thorax, in members of the family Diazonidae
and in Aplousobranchia a true posterior abdomen exists, separating the zooids into two distinct parts (Kott, 1990). This division
of the body allows some Aplousobranchia species to lose/degenerate their thorax when the environmental conditions are harsh and
regenerate it when conditions improve (Brien, 1930; Turon, 1992).
Interestingly, similar thorax regeneration has been mentioned in R.
cloneyi (Vazquez and Young, 1996).
Previous taxonomic classifications grouped together the genera
Ciona, Rhopalaea and Diazona within the family Cionidae, order
Phlebobranchia (Monniot et al., 1991) based on their similar branchial sac characteristics (e.g., the presence of inner longitudinal
branchial vessels). Kott (1990), on the contrary, placed Ciona as
the only known genus of the family Cionidae Lahille, 1887, and
included Rhopalaea, and Diazona within the family Diazonidae
Seeliger, 1906, order Aplousobranchia. Consequently, according
to Kott (1990, 2005), the loss of longitudinal branchial vessels in
colonial aplousobranchs and stolidobranchs is a convergent
adaptation associated with the reduction in zooid size and does
not indicate a phylogenetic affinity.
A broad phylogenetic analysis of Aplousobranchia relationships
based on 47 morphological characters has left unresolved the position of Rhopalaea and Diazona, (Moreno and Rocha, 2008). In this
study, the later genera are early offshoots in the ascidian tree,
while Ciona is placed together with Phlebobranchia and Stolidobranchia representatives. This led Moreno and Rocha (2008) to
exclude Cionidae and Diazonidae from the Aplousobranchia.
Molecular phylogenetic analyses have not yet resolved the positioning of Diazonidae and Cionidae. The phylogenetic position of
Diazonidae has only been investigated based on COI sequences
(Turon and López-Legentil, 2004). In this study, Ciona is placed as
a sister clade of Aplousobranchia and Rhopalaea branches within
Aplousobranchia, suggesting that Cionidae and Diazonidae are
members of the order Aplousobranchia. However, the position of
Rhopalaea has not been corroborated by high support values except
when using the Bayesian criteria. Since Bayesian posterior probabilities have been shown to overestimate branch supports
(Douady et al., 2003; Erixon et al., 2003), the study of Turon and
López-Legentil (2004) does not exclude the possibility that Diazonidae could be the earliest diverging Aplousobranchia family.
Additionally, analysis of complete mt sequences (Rubinstein
et al., 2013) and 18S rDNA sequences (Tsagkogeorga et al., 2009)
did not confirm the grouping of Cionidae with Aplousobranchia.
In these studies, Cionidae is either the sister clade of Phlebobranchia + Thaliacea or the sister clade of Thaliacea. Thus, morphological and molecular data produce contrasting results regarding
the phylogenetic position of the families Diazonidae and Cionidae,
which still remains uncertain.
Here, we investigate the phylogenetic position of a solitary
member of the Diazonidae family, Rhopalaea idoneta (Shenkar,
2012), using mitochondrial protein-coding genes. In addition, we
also examined the regenerative abilities of this species. R. idoneta
is a highly abundant species on the coral reefs of Eilat, Red Sea,
Israel, found at 5–60 m depth. Field surveys have revealed that it
can reach a density of 3 individuals/m2 at 30 m depth (Koplovitz
and Shenkar, 2014). On the coral reefs only the thorax is visible,
while the abdomen, which contains the digestive system and
gonads, remains completely embedded in the substrate (Fig. 1A).
Phylogenies based on complete mitochondrial genome sequences
have been shown to successfully resolve deep-level relationships
among various metazoan groups, including ascidians (Rubinstein
et al., 2013; Singh et al., 2009). Consequently, we sequenced
the complete mitochondrial genome of R. idoneta and reconstructed the tunicate phylogeny using the 13 mitochondrial
protein-coding genes.
Similar to colonial aplousobranch species, some members of the
Rhopalaea genus are known to possess the ability to regenerate
their thorax (Monniot and Monniot, 2001). However, this
phenomenon has not been documented to date or studied in depth.
We therefore conducted field experiments to further investigate
this feature in R. idoneta.
2. Material and methods
2.1. Sampling, DNA sequencing and annotations
On February 9th, 2014, five individuals of Rhopalaea idoneta
were sampled at 33 m depth using SCUBA. The specimens were
collected from the natural coral reef in front of the
Inter-University Institute in Eilat (29°300 5.6900 N, 34°550 2.9200 E),
under permit number 40201/2014, Israel Nature and Parks
Authority. They were preserved in 99% ethanol and deposited at
the Steinhardt Museum of Natural History at Tel Aviv University,
voucher number SMNHTAU-AS25947.
Total DNA (gDNA) was extracted from a small portion of siphonal tissue following the protocol of Fulton et al. (1995). To reduce
the sequencing cost, the DNA of Rhopalaea was mixed with DNA of
non-tunicate species (which are part of another work). The mixed
gDNA was sent for library formation and gDNA sequencing to the
Technion Genome Center (Haifa, Israel). A total of 20,000,000
paired reads (each 100 bp long) were obtained with the Illumina
Hiseq 2000 sequencer. Adapter sequences were removed using
Cutadapt (Martin, 2011). To assemble the complete mitochondrial
genome, we followed the approach of Rubinstein et al. (2013) and
used the transcriptome assembler SOAPdenovo-Trans (Xie et al.,
2014), rather than a genomic assembler, with a k-mer length of
64. Indeed, genomic assemblers which assume uniform read coverage often fail to assemble complete mitochondrial contigs from
mixed gDNA samples (Rubinstein et al., 2013). A Blast search was
conducted to identify the mitogenome contig using the COI
sequence of Rhopalaea neapolitana (AY600983) as query. A coverage analysis was then performed with Geneious Pro (Version
6.1.7, Biomatters, Auckland, New Zealand) using paired-reads. This
allowed us to confirm that the contig obtained was circular and to
verify the quality of the assembly. The mean coverage was 100
(SD 19; minimum 35, maximum 162).
The mitochondrial sequence was annotated following the
methods detailed in Rubinstein et al. (2013). The mtDNA sequence
of R. idoneta was deposited at the EMBL-EBI European Nucleotide
Archive under accession number LN877970.
2.2. Phylogenetic analyses
The 13 protein-coding genes of the 35 tunicate mitochondrial
genomes available in GenBank (July, 2015) (Supplementary
Table S1), and of 18 representative outgroups (Singh et al., 2009),
were aligned at the amino acid level using MAFFT under the
L-INS-i refinement strategy (Katoh and Standley, 2013). The
webserver Guidance (Penn et al., 2010) was used to remove
positions with a low confidence score (i.e., below 0.93), as well
as positions present in less than 25% of the species. The concatenated alignment of the 13 mitochondrial proteins is available as
Supplementary File S2.
Bayesian phylogenetic reconstructions were performed with
Phylobayes 3.3b (Lartillot et al., 2009) under the CAT + GTR + C
mixture model. Three Markov Monte Carlo chains (MCMC) were
run for 51,000 cycles and sampled every 10 cycles. After a manual
examination of the trace files produced by Phylobayes, the first
N. Shenkar et al. / Molecular Phylogenetics and Evolution 100 (2016) 51–56
53
Fig. 1. Mitochondrial evolution. A. Rhopalaea idoneta. B. Tunicate phylogenetic relationships. Red branches indicate colonial taxa. The Thaliacea Doliolum, whose life cycle
alternates between colonial and solitary forms, is indicated by a red and black branch. The phylogeny was inferred from the concatenation of the 13 mitochondrial proteins
(3131 amino-acids and 54 taxon). Bayesian consensus tree of 3 independent MCMC runs obtained using the CAT + GTR + C mixture model. Most of the branches received
maximal support (PP = 1.0). Consequently, branch support values are only indicated for nodes with PP below 0.99. Accession numbers are provided in Table S1. C.
Organization of the Rhopalaea idoneta mt genome. The tRNA genes are marked in black by their one-letter code, except for G(a), Gly(AGR); G(g), Gly(GGN); L(u), Leu(UUR); L
(c), Leu(CUN); M(c), Met(CAU); M(u), Met(UAU); S(a), Ser(AGY); and S(u), Ser(UCN). All genes are transcribed clockwise. rRNAs are in red. ATP synthase genes are in orange.
NADH dehydrogenase genes (complex I) are in blue. The cytochrome b (complex III) is in purple. Cytochrome c oxidase genes (complex VI) are in green. Non-coding regions
are marked in white. Species names as in Supplementary Table S1, with Ciona intestinalis former Ciona intestinalis type B, and Ciona robusta former Ciona intestinalis type A
(Brunetti et al., 2015). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
1000 trees were discarded (burn-in). The maximum and average
differences, observed at the end of the run were 0.05 and 0.001,
respectively, which indicate a correct convergence. We also verified
that the parameters ‘‘rel_diff” and ‘‘effsize” were lower than 0.3 and
higher than 50, respectively, which indicates an acceptable run.
2.3. Regeneration experiments
In order to provide additional physiological traits supporting
the aplousobranch affinity of Rhopalaea idoneta, we investigated
whether individuals are capable of regenerating a complete new
thorax following its artificial removal. A field experiment was carried out at 30–35 m depth in front of the Interuniversity Institute
in Eilat, (29°300 06.200 N, 34°550 01.700 E) between February and June
2014, in which six individuals of uniform size (thorax 5 cm long)
were measured in the field, photographed, and marked with plastic
coated metal cords. Subsequently, we manually removed the thorax from each individual, leaving the abdomen buried in crevices
or coral branches (Shenkar, 2012). The experiment site was revisited two and three months post-thorax-removal, at which time
54
N. Shenkar et al. / Molecular Phylogenetics and Evolution 100 (2016) 51–56
the individuals were visually examined and photographed. Following the three-month study, we repeated the experiment on the
same individuals. The thorax was again removed manually, and
the marked individuals were revisited one month later (June 2014).
In order to follow the regeneration progress on a daily basis, in
May 2014 we transferred one individual with its associated substrate to a controlled water table at the Interuniversity Institute,
and repeated the thorax-removal procedure. The regeneration progress of this individual was followed by photographing on a daily
basis for two weeks. The experiment was repeated on the same
individual in November 2014. Following thorax removal, siphon
development was monitored for ten days.
3. Results
3.1. Mitochondrial genome structure
The mitochondrial genome of R. idoneta was found to be
14,939 bp long, which is within the range of other tunicate genomes
(Fig. 1C). It possesses the regular tunicate gene content: 13 proteincoding genes, two ribosomal RNA genes, and a total of 24 tRNAs
(Supplementary Fig. S1). In agreement with other tunicate mt genomes described (Gissi et al., 2010; Rubinstein et al., 2013), all genes
share the same strand orientation. The gene order is extremely rearranged even when compared to closely related species. Indeed, R.
idoneta possesses a novel gene order which shares only two gene
pairs with most of the remaining aplousobranchs (i.e., trnS(UCN) –
cox3 and trnA-atp6 are shared with all other aplousobranchs, except
Didemnum vexillum and Diplosoma listerianum, respectively).
3.2. Molecular phylogeny
The phylogenetic tree (Fig. 1B) reconstructed under the Bayesian CAT model is well supported except for four nodes with posterior probabilities (PP) below 0.9. Deuterostome relationships
agree with the findings of Rubinstein et al. (2013). Among tunicates, the addition of new sequences did not modify the relationships within the Stolidobranchia, in which the Styelidae
(Botryllus, Botrylloides, Polycarpa, and Styela) remain monophyletic
and the Pyuridae paraphyletic, as in Rubinstein et al. (2013). The
stolidobranch phylogeny is thus very stable and presents maximal
support values (PP = 1.0), except for some relationships among
species of the genera Botryllus and Botrylloides. In contrast, the relationships within the monophyletic clade grouping Aplousobranchia, Phlebobranchia, and Thaliacea (PP = 1.0) are modified
compared to Rubinstein et al. (2013): (a) Thaliacea are now placed,
with moderate support (PP = 0.8), as sister clade of Aplousobranchia + Phlebobranchia, and not nested within Phlebobranchia;
(b) Phlebobranchia are paraphyletic since Ciona is placed as a sister
group of Aplousobranchia (PP = 0.91), whereas in Rubinstein et al.
(2013) Ciona is the sister lineage of the Phlebobranchia + Thaliacea
clade (although with moderate support, i.e., PP = 0.79); (c)
Aplousobranchia are monophyletic (PP = 1.0) and include the solitary Rhopalaea (Diazonidae). Moreover, Rhopalaea is not of the first
diverging Aplousobranchia but is a sister clade of Aplidium (Polyclinidae) (PP = 1.0), while the colonial Clavelina (Clavelinidae) are
the first aplousobranchs to diverge (PP = 1.0).
3.3. Regeneration experiments
Two months following thorax removal, five out of the six
marked individuals were found with a fully developed thorax,
reaching its original size. During the second thorax-removal round,
three out of the five individuals were found with a complete and
functioning thorax, one month later. The daily monitoring of the
regenerating individual in the lab revealed that new siphons were
visible two days after thorax removal, and a fully developed functional thorax, the same size as prior to removal, was visible after
only seven days (Fig. 2). The second removal trial, which was conducted after six months on the same individual in the water table,
yielded the same results. Throughout the entire monitoring period
the regenerating individual exhibited completely healthy and
functioning behavior (determined visually by initiating a siphonal
contraction response).
4. Discussion
The current study presents a solid support for a positioning of
the Diazonidae family, here represented by the solitary species Rhopalaea idoneta, as a member of the Aplousobranchia order. The
mitochondrial phylogeny (Fig. 1B) reveals that R. idoneta is not an
early diverging member of the Aplousobranchia, as suggested by
Kott (1989), but has a more internal position and is a sister clade
of Aplidium. Our study thus supports the scenario of an adaptation
to a solitary lifestyle from an aplousobranch colonial ancestor. It has
been demonstrated that a colonial life-style has evolved several
time independently in Stolidobranchia and Phlebobranchia, in particular within Styelidae (Pérez-Portela et al., 2009). Though Zeng
et al. (2006) originally suggested the monophyly of colonial Styelidae based on a restricted stolidobranch taxon sample. In the
Aplousobranchia sensu Kott (1990) we are able to hypothesize that
the coloniality is an ancestral trait which originally evolved from a
Ciona-like solitary ancestor and then reverted to the solitary lifestyle in at least one lineage. Future analysis of colonial representatives of Diazonidae, such as a Diazona species, is needed in order to
ascertain the monophyletic character of Diazonidae and to definitively confirm its nested position in the Aplousobranchia.
Most aplousobranch species are capable of whole thorax
regeneration by the epicardium (Kott, 2005). The epicardium, or
epicardial sacs, are paired endodermal sacs that evaginate from
the postero-ventral part of the pharynx during embryogenesis
(Berrill, 1951; Kott, 1990). In the Phlebobranchia and Stolidobranchia the epicardium has an excretory role. In the Aplousobranchia and Ciona, however, the epicardium is a source of stem
cells, allowing rapid regeneration and budding (Berrill, 1951;
Jeffery, 2015). Similarly to the broad regenerative abilities of Ciona
intestinalis (Jeffery, 2015), our field experiments confirmed that
R. idoneta possess the ability to completely regenerate its thorax
within only a few days (Fig. 2), presumably by stem cells originating from the epicardium, which extends along the embedded abdomen. Other solitary ascidians from the orders Stolidobranchia and
Phlebobranchia are not capable of regenerating a full thorax, as
their body is not divided into two distinct body parts as in
Rhopalaea. Thus, in Stolidobranchia and Phlebobranchia, the thorax
is not homologous to the Aplousobranchia thorax since it does not
contain the same organs, therefore it cannot be fully removed
without damaging the viscera. In the tropical habitat of R. idoneta,
the rapid regeneration of such a gelatinous thorax may provide an
effective mechanism against fish predation, and may further
explain the relatively high abundance of this species in the
Red-Sea coral reefs (Koplovitz and Shenkar, 2014).
The increase in colonial integration is associated, in ascidians,
with a decrease in zooid size (Ryaland and Warner, 1986). Thus,
many aplousobranch species are characterized by small and
simplified zooids. In the case of the genus Rhopalaea, adopting a
solitary life-style may have driven the evolution of a large
thorax size in order to increase filtration rates resulting in the
re-acquisition of a ‘‘phlebobranch trait” as longitudinal vessels.
For example, R. idoneta, R. macrothorax, and R. crassa are all tropical
species characterized by a relatively much larger thorax (5–8 cm
long) than those of colonial Diazona (10–15 mm long, Tokioka,
1953; Shenkar, 2012).
N. Shenkar et al. / Molecular Phylogenetics and Evolution 100 (2016) 51–56
55
Fig. 2. Regeneration in Rhopalaea idoneta. (a) A complete individual before thorax removal. (b) Immediately after thorax removal. Black circle denotes the hole from which the
thorax emerged. (c) Newly developing thorax three days post-removal. (d) New and complete thorax nine days post-removal. os – oral siphon; as – atrial siphon, scale bar
1 cm.
The most studied ascidian, Ciona, which has long been suggested as the most primitive extant ascidian (Kott, 1989), is currently classified as a phlebobranch (Shenkar et al., 2015).
However, our present findings suggest that Ciona is in fact a sister
clade of Aplousobranchia, as also suggested in the past based both
on molecular data (Turon and López-Legentil, 2004) and morphological characters (Kott, 1990, 2005). However, it should be
stressed that the relationships among early-diverging Phlebobranchia and Aplousobranchia are unstable, as evident by the
low support values and the topological changes observed between
studies (see Rubinstein et al., 2013, Fig. 4, for comparison). Thus,
the sequencing of additional aplousobranch species, and in particular the sequencing of additional genera belonging to the family
Cionidae, as well as the reevaluation of Cionidae morphological
characters, may assist in accurately defining its phylogenetic
position.
In sum, our results support the assigning of Diazonidae to the
Aplousobranchia, and the loss of a colonial lifestyle in Rhopalaea,
since this species did not diverge early among the Aplousobranchia. The unique phylogenetic position of this large solitary
Diazonidae species within a generally colonial clade, together with
its wide regenerative ability, makes Rhopalaea an interesting model
by which to extend our knowledge on the evolution of regeneration and budding in tunicates.
Acknowledgments
We would like to thank Dayana Yahalomi for performing the
data assembly, and The Steinhardt Museum of Natural History
and National Research Center staff for their support. This research
was supported by the German-Israeli Foundation Grant number
I-2325-1113.13/2012 to N.S., the Israel Taxonomy Initiative Grant
to N.S and G.K. and The Israel Science Foundation Grant number
161/15 to DH.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2016.04.
001.
References
Berrill, N.J., 1951. Regeneration and budding in tunicates. Biol. Rev. 26, 456–475.
Brien, P., 1930. Contribution à l’étude de la régénération naturelle and
expérimentale chez les Clavelinidae. Soc. Roy. Zool. Belg. Ann. LXI, 19–112.
Brunetti, R., Gissi, C., Pennati, R., Caicci, F., Gasparini, F., Manni, L., 2015.
Morphological evidence that the molecularly determined Ciona intestinalis
type A and type B are different species: Ciona robusta and Ciona intestinalis. J.
Zool. Syst. Evol. Res. 53, 186–193.
Dehal, P., Satou, Y., Campbell, R.K., Chapman, J., Degnan, B., De Tomaso, A., Davidson,
B., Di Gregorio, A., Gelpke, M., Goodstein, D.M., Harafuji, N., Hastings, K.E., Ho, I.,
Hotta, K., Huang, W., Kawashima, T., Lemaire, P., Martinez, D., Meinertzhagen, I.
A., Necula, S., Nonaka, M., Putnam, N., Rash, S., Saiga, H., Satake, M., Terry, A.,
Yamada, L., Wang, H.G., Awazu, S., Azumi, K., Boore, J., Branno, M., Chin-Bow, S.,
DeSantis, R., Doyle, S., Francino, P., Keys, D.N., Haga, S., Hayashi, H., Hino, K.,
Imai, K.S., Inaba, K., Kano, S., Kobayashi, K., Kobayashi, M., Lee, B.I., Makabe, K.
W., Manohar, C., Matassi, G., Medina, M., Mochizuki, Y., Mount, S., Morishita, T.,
Miura, S., Nakayama, A., Nishizaka, S., Nomoto, H., Ohta, F., Oishi, K., Rigoutsos,
I., Sano, M., Sasaki, A., Sasakura, Y., Shoguchi, E., Shin-I, T., Spagnuolo, A.,
Stainier, D., Suzuki, M.M., Tassy, O., Takatori, N., Tokuoka, M., Yagi, K., Yoshizaki,
F., Wada, S., Zhang, C., Hyatt, P.D., Larimer, F., Detter, C., Doggett, N., Glavina, T.,
Hawkins, T., Richardson, P., Lucas, S., Kohara, Y., Levine, M., Satoh, N., Rokhsar, D.
S., 2002. The draft genome of Ciona intestinalis: insights into chordate and
vertebrate origins. Science 298, 2157–2167.
Douady, C.J., Delsuc, F., Boucher, Y., Doolittle, W.F., Douzery, E.J.P., 2003.
Comparison of Bayesian and maximum likelihood bootstrap measures of
phylogenetic reliability. Mol. Biol. Evol. 20, 248–254.
Erixon, P., Svennblad, B., Britton, T., Oxelman, B., 2003. Reliability of Bayesian
posterior probabilities and bootstrap frequencies in phylogenetics. Syst. Biol.
52, 665–673.
Fulton, T.M., Chunwongse, J., Tanksley, S.D., 1995. Microprep protocol for extraction
of DNA from tomato and other herbaceous plants. Plant. Mol. Biol. Rep. 13, 207–
209.
Gissi, C., Pesole, G., Mastrototaro, F., Iannelli, F., Guida, V., Griggio, F., 2010. Hypervariability of ascidian mitochondrial gene order: exposing the myth of
deuterostome organelle genome stability. Mol. Biol. Evol. 27, 211–215.
56
N. Shenkar et al. / Molecular Phylogenetics and Evolution 100 (2016) 51–56
Hawkins, C.J., Kott, P., Parry, D.L., 1983. Vanadium content and oxidation state
related to ascidian phylogeny. Comp. Biochem. Phys. 76B, 555–558.
Jeffery, W.R., 2015. Closing the wounds: one hundred and twenty five years of
regenerative biology in the ascidian Ciona intestinalis. Genesis 53, 48–65.
Katoh, K., Standley, D.M., 2013. MAFFT multiple sequence alignment software version
7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780.
Koplovitz, G., Shenkar, N., 2014. The Biodiversity of the Class Ascidiacea in the Gulf
of Eilat (Aqaba). Israel Taxonomy Initiative Report <http://taxonomy.tau.ac.il/
eng/content/biodiversity-survey-results>.
Kott, P., 1989. Form and function in the Ascidiacea. Bull. Mar. Sci. 45, 253–276.
Kott, P., 1990. The Australian Ascidiacea. Part 2, Aplousobranchia (1). Mem. Qld.
Mus. 29, 1–299.
Kott, P., 2005. Catalogue of Tunicata in Australian Waters. Australian Biological
Resources Study, Canberra, p. 301.
Lahille, F., 1887. Sur la classification des tuniciers. C. R. Hebd. Seanc. Acad. Sci., Paris
102, 1573–1575.
Lartillot, N., Lepage, T., Blanquart, S., 2009. PhyloBayes 3: a Bayesian software
package for phylogenetic reconstruction and molecular dating. Bioinformatics
25, 2286–2288.
Martin, M., 2011. Cutadapt removes adapter sequences from high-throughput
sequencing reads. EMBnet.journal 17, 10–12.
Monniot, C., 1991. Ascidies de Nouvelle-Calédonie VIII. Phlébobranches (suite). Bull.
Mus. Nat. His. Nat. 4, 12, 3–4.
Monniot, F., Monniot, C., 2001. Ascidians from the tropical western Pacific.
Zoosystema 23 (2), 201–376.
Monniot, C., Monniot, F., Laboute, P., 1991. Coral Reef Ascidians of New Caledonia
(No. 30). IRD Editions.
Moreno, T.R., Rocha, R.M., 2008. Phylogeny of the Aplousobranchia (Tunicata:
Ascidiacea). Rev. Bras. Zool. 25, 269–298.
Penn, O., Privman, E., Ashkenazy, H., Landan, G., Graur, D., Pupko, T., 2010.
GUIDANCE: a web server for assessing alignment confidence scores. Nucleic
Acids Res. 38, W23–W28.
Pérez-Portela, R., Bishop, J.D.D., Davis, A.R., Turon, X., 2009. Phylogeny of the
families Pyuridae and Styelidae (Stolidobranchiata, Ascidiacea) inferred from
mitochondrial and nuclear DNA sequences. Mol. Phylo. Evol 50 (3), 560–570.
Rubinstein, N.D., Feldstein, T., Shenkar, N., Botero-Castro, F., Griggio, F.,
Mastrototaro, F., Delsuc, F., Douzery, E.J.P., Gissi, C., Huchon, D., 2013. Deep
sequencing of mixed total DNA without barcodes allows efficient assembly of
highly plastic ascidian mitochondrial genomes. Genome Biol. Evol. 5, 1185–
1199.
Ryaland, J.S., Warner, G.F., 1986. Growth and form in modular animals: ideas on the
size and arrangement of zooids. Philos. Ttans. R. Soc. B 313, 53–76.
Seeliger, O., 1906. Appendicularien und ascidian (Tunicata, Manteltierie). In: Bronn,
H.G. (Ed.), Klassen und Ordnungen des Tier-Riechs, vol. 3. C.F. Winter, Leipzig
(pp. 26–80, 385–1280, Continued by Hartmeyer 1909–1911).
Shenkar, N., 2012. A new species of the genus Rhopalaea (Class: Ascidiacea) from the
Red Sea. Zootaxa 3599, 51–58.
Shenkar, N., Gittenberger, A., Lambert, G., Rius, M., Rocha, R.M., Swalla, B.J., Turon,
X., 2015. Ascidiacea World Database <http://www.marinespecies.org/
ascidiacea> (accessed September 2nd 2015).
Singh, T.R., Tsagkogeorga, G., Delsuc, F., Blanquart, S., Shenkar, N., Loya, Y., Douzery,
E.J.P., Huchon, D., 2009. Tunicate mitogenomics and phylogenetics: peculiarities
of the Herdmania momus mitochondrial genome and support for the new
chordate phylogeny. BMC Genom. 10 (1), 534.
Tokioka, T., 1953. Ascidians of Sagami Bay. Iwanami Shoten, Tokyo, pp. 247–248.
Tsagkogeorga, G., Turon, X., Hopcroft, R.R., Tilak, M.-K., Feldstein, T., Shenkar, N.,
Loya, Y., Huchon, D., Douzery, E.J.P., Delsuc, F., 2009. An updated 18S rRNA
phylogeny of tunicates based on mixture and secondary structure models. BMC
Evol. Biol. 9, 187.
Turon, X., 1992. Periods of non-feeding in Polysyncraton lacazei (Ascidiacea:
Didemnidae): a rejuvenative process? Mar. Biol. 112 (4), 647–655.
Turon, X., López-Legentil, S., 2004. Ascidian molecular phylogeny inferred from
mtDNA data with emphasis on the Aplousobranchiata. Mol. Phylogenet. Evol.
33 (2), 309–320.
Vazquez, E., Young, C.M., 1996. Rhopalaea cloneyi (Ascidiacea, Cionidae), a new
deep-water ascidian from the coastal waters of British Columbia. J. Zool. 238,
599–610.
Voskoboynik, A., Neff, N.F., Sahoo, D., Newman, A.M., Pushkarev, D., Koh, W., Quake,
S.R., 2013. The genome sequence of the colonial chordate, Botryllus schlosseri.
Elife 2, e00569.
Xie, Y., Wu, G., Tang, J., Luo, R., Patterson, J., Liu, S., Huang, W., He, G., Gu, S., Li, S.,
Zhou, X., Lam, T.W., Li, Y., Xu, X., Wong, G.K., Wang, J., 2014. SOAPdenovo-Trans:
de novo transcriptome assembly with short RNA-Seq reads. Bioinformatics 30,
1660–1666.
Zeng, L., Jacobs, M.W., Swalla, B.J., 2006. Coloniality has evolved once in
Stolidobranch Ascidians. Integr. Comp. Biol. 46, 255–268.